Devices for biological sample collection and analysis and methods of use thereof

ABSTRACT

In some embodiments, the present invention is a device, including: a swab including an absorptive component attached to a stem, an extraction chamber configured to receive the swab and position the absorptive component of the swab configured to be in fluid communication with an extraction reagent, a test strip configured to be brought in fluid communication with the extraction reagent following extraction of the analyte from the biological sample, including: a sample receiving portion configured to accept a sample, a site on the strip where the analyte-specific labeled reagent has been incorporated, such reagent configured to bind the analyte from the biological sample, a capture portion configured to receive: the analyte from the biological sample and the analyte-specific labeled reagent so as to result in displaying a positive or negative result at the completion of the assay, and an adsorbent pad attached to the test strip.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/827,237, filed Mar. 23, 2020, which is a continuation of Ser. No.15/548,971, filed Aug. 4, 2017, which is 371 National Phase Applicationof International Application No. PCT/IB2016/000296, filed Feb. 17, 2016,which claims priority of U.S. Provisional Application No. 62/117,211;filed Feb. 17, 2015, which are incorporated herein by reference in theirentirety for all purposes.

TECHNICAL FIELD

In some embodiments, the present instant invention is related todetection tests, which are diagnostic tests which can be used inassisting in the diagnosis of bacterial pharyngitis caused by group Astreptococci (GAS) and methods of use thereof.

BACKGROUND

Group A Streptococcus (Streptococcus pyogenes) typically causes acuteupper respiratory tract infection. Early diagnosis and treatment ofGroup A Streptococcal pharyngitis is critical for reducing the severityof symptoms and complications, such as rheumatic fever andglomerulonephritis, and preventing the rare but possible occurrence ofdeath—of the 9,000-11,500 cases of invasive disease (3.2 to 3.9/100,000population) that occur each year in the United States alone, 10%-15%result in death. Typical diagnostic tests used for assaying Group AStreptococcus antigens require the mixing of equal to or more than tworeagents and/or use laborious test-specific protocols.

SUMMARY OF INVENTION

In some embodiments, the present invention is a device, including:

-   -   a swab including an absorptive component attached to a stem,        where the absorptive component includes a non-volatile reagent,        an extraction chamber configured to receive the swab and        position the absorptive component of the swab configured to be        in fluid communication with an extraction reagent,        -   where the extraction chamber is filled with an extraction            reagent, a test strip configured to be brought in fluid            communication with the extraction reagent following            extraction of the analyte from the biological sample,            including:    -   a sample receiving portion configured to accept a sample,        usually a liquid sample, and permit the movement of any analyte        in the liquid through the test strip via, e.g., but not limited        to, capillary action,    -   a site on the strip where the analyte-specific labeled reagent        has been incorporated, such reagent configured to bind the        analyte from the biological sample, and includes any molecule        that binds the analyte specifically and with high affinity and        is further labeled with a label that allows its detection,    -   a capture portion configured to receive: the analyte from the        biological sample and the analyte-specific labeled reagent so as        to result in displaying a positive or negative result at the        completion of the assay, and an adsorbent pad attached to the        distal end of the test strip and configured to bind to an excess        extraction reagent thereby allowing flow across the test strip.

In some embodiments, the analyte is Streptococcus Group A CarbohydrateAntigen. In some embodiments, the extraction reagent is a nitrite salt.In some embodiments, the extraction reagent is 0.2-5M nitrite saltsolution. In some embodiments, the non-volatile reagent is acidic.

In some embodiments, the swab and test strip are configured to be influid communication, so as to result in capillary flow from theabsorptive component of the swab to a sample receiving portion of thetest strip. In some embodiments, the stem may be solid, porous, or anycombination thereof. In some embodiments, the stem is configured toprovide: mechanical support for the absorptive component of the swab,and capillary flow through a porous core of the stem. In someembodiments, the absorptive component of the swab is composed of afiber, foam of polymeric material, absorbent material, or anycombination thereof. In some embodiments, the acidic non-volatilereagent is deposited at the absorptive component of the swab by:spraying, dipping, or dispersing the absorptive component of the swab ina solvent containing the acidic non-volatile reagent, and evaporatingthe solvent.

In some embodiments, the amount of the acidic non-volatile reagent isbetween 2 and 800 micromoles. In some embodiments, the acidicnon-volatile reagent is soluble in the extraction reagent. In someembodiments, the acidic non-volatile reagent is insoluble in theextraction reagent. In some embodiments, the acidic non-volatile reagentis configured to exchange protons with the extraction reagent. In someembodiments, the insoluble acidic non-volatile reagent is deposited onthe swab in a region configured to attach to the absorptive component,and where the insoluble acidic non-volatile reagent is deposited on theswab by coating with a membrane or film made of a polymeric materialprior to the application of the absorptive component, so as to result inavoiding direct contact between the acid and the subject. In someembodiments, the acidic non-volatile reagent is organic. In someembodiments, the acidic non-volatile reagent is inorganic. In someembodiments, the device includes: a first structural componentconfigured to attach the absorptive component of the swab by the stem ofthe swab, a second structural component configured to house the teststrip, where the absorptive component and a sample receiving portion ofthe test strip are in liquid communication when joining the firststructural component and the second structural component.

In some embodiments, the present invention is a method, including:

-   -   (i) collecting a biological sample of a subject by use of a swab        including a first, non-volatile reagent;    -   (ii) transferring the swab to a chamber containing an extraction        reagent, so as to allow the non-volatile reagent to react with        the extraction reagent and result in generating an extraction        solution;    -   (iii) extracting an analyte from the biological sample of the        subject by the extraction solution;    -   (iv) contacting the extraction solution containing the extracted        analyte with a lateral flow immunochromatographic assay device        including a labeled analyte-specific indicator; and    -   (v) determining a presence or absence of the extracted analyte        in the biological sample of the subject, where the binding of        the analyte- to a capture reagent immobilizes the labeled        analyte-indicator complex on the lateral flow        immunochromatographic assay device, and where the capture        reagent is configured to specifically bind to the analyte.

In some embodiments, the present invention is a method, including:

-   -   (i) collecting a biological sample of a subject by an absorptive        component of a swab, where the absorptive component of the swab        is configured to be in physical contact with a sample receiving        portion of a lateral flow immunochromatographic assay test strip        including a labeled analyte-specific indicator;    -   (ii) transferring the absorptive component of the swab to a        chamber containing an extraction reagent,    -   where an extraction solution is formed in-situ by the reaction        of the extraction reagent with the second non-volatile reagent        incorporated in the absorptive component of the swab, and where        the extraction solution is configured to extract the biological        sample of the subject;    -   (iii) extracting an analyte from the biological sample;    -   (iv) determining the presence or absence of the extracted        analyte in the biological sample of the subject by measuring a        signal generated by a binding of a labeled analyte-indicator        complex to a capture reagent,    -   where the capture reagent is immobilized on the lateral flow        immunochromatographic assay device, and    -   where the capture reagent is configured to specifically bind to        the analyte.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further explained with reference to theattached drawings, wherein like structures are referred to by likenumerals throughout the several views. The drawings shown are notnecessarily to scale, with emphasis instead generally being placed uponillustrating the principles of the present invention. Further, somefeatures may be exaggerated to show details of particular components.

FIGS. 1A-B shows an embodiment of the device of the present invention,which is a side view of a typical swab, comprised of stem and tip (i.e.,the absorptive component), indicating reagent incorporation sites. FIG.1A depicts reagent application by coating the swab's stem; FIG. 1Bdepicts reagent incorporation into the swab tip's area that is away fromthe sample collection zone.

FIG. 2 shows an embodiment of the device of the present invention, whichis a side view of a diagnostic kit that includes a swab, extractionchamber and test strip, indicating the relative positions of each of thecomponents.

FIG. 3 shows an embodiment of the device of the present invention, whichis a perspective view of a cross section of a diagnostic kit thatincludes an extraction chamber and an integrated swab-test strip devicethat connects the back end of the swab tip to the sample pad of the teststrip allowing for capillary liquid communication between both media;the integrated device is enclosed within a cylindrical shaped casingthat also acts as a handle for the integrated device, replacing theswab's stem for grip and allowing sample collection and analysis withinthe same integrated device.

FIGS. 4A-C shows detail of a preferred embodiment of the integratedswab-test strip device of the present invention, providing physicalcontact between the swab and test strip, thereby allowing capillaryliquid communication between the two; FIG. 4A shows a perspective viewof the integrated device, displaying all major components; FIG. 4Bprovides additional detail for swab tip mechanical immobilization withinthe integrated device; FIG. 4C shows the fully assembled integrateddevice.

The figures constitute a part of this specification and includeillustrative embodiments of the present invention and illustrate variousobjects and features thereof. Further, the figures are not necessarilyto scale, some features may be exaggerated to show details of particularcomponents. In addition, any measurements, specifications and the likeshown in the figures are intended to be illustrative, and notrestrictive. Therefore, specific structural and functional detailsdisclosed herein are not to be interpreted as limiting, but merely as arepresentative basis for teaching one skilled in the art to variouslyemploy the present invention.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

Among those benefits and improvements that have been disclosed, otherobjects and advantages of this invention will become apparent from thefollowing description taken in conjunction with the accompanyingfigures. Detailed embodiments of the present invention are disclosedherein; however, it is to be understood that the disclosed embodimentsare merely illustrative of the invention that may be embodied in variousforms. In addition, each of the examples given in connection with thevarious embodiments of the invention is intended to be illustrative, andnot restrictive.

Throughout the specification and claims, the following terms take themeanings explicitly associated herein, unless the context clearlydictates otherwise. The phrases “in one embodiment” and “in someembodiments” as used herein do not necessarily refer to the sameembodiment(s), though it may. Furthermore, the phrases “in anotherembodiment” and “in some other embodiments” as used herein do notnecessarily refer to a different embodiment, although it may. Thus, asdescribed below, various embodiments of the invention may be readilycombined, without departing from the scope or spirit of the invention.

In addition, as used herein, the term “or” is an inclusive “or”operator, and is equivalent to the term “and/or,” unless the contextclearly dictates otherwise. The term “based on” is not exclusive andallows for being based on additional factors not described, unless thecontext clearly dictates otherwise. In addition, throughout thespecification, the meaning of “a,” “an,” and “the” include pluralreferences. The meaning of “in” includes “in” and “on.”

As used herein, a “lateral flow test” refer to an immunochromatographicdevice intended to detect the presence (or absence) of a target analytein sample (matrix) without the need for specialized and costlyequipment. Typically, these tests are used for medical diagnosticseither for home testing, point of care testing, or laboratory use.

As used herein, a “sample receiving portion” or a “fluid receivingportion” refers to a portion of a test strip configured to accept asample, usually a liquid/fluid sample, and permit the movement of anyanalyte in the liquid/fluid through the test strip via, e.g., but notlimited to, capillary action.

As used herein, an “analyte-specific labeled reagent” or a “labeledanalyte-specific indicator” are synonymous and refer to what is commonlyknown in the diagnostic literature as a “conjugate”—a dried form ofbio-active particles in a salt-sugar matrix that contains everything toguarantee an optimized chemical reaction between the target molecule(e.g., the analyte) and its chemical partner (e.g., antibody) that hasbeen immobilized on the particle's surface.

As used herein, a “capture portion” or “capture zone” refers to aportion of a test strip where analyte-reagent complexes bind to acapture reagent (e.g., but not limited to, an analyte-specificantibody).

As used herein, “sorptivity” refers to a measure of the capacity of themedium to absorb or desorb liquid by capillarity.

In some embodiments, the device of the present invention is configuredto measure the presence or absence of Streptococcus Group A antigen in abiological sample. In some embodiments, the device comprises: (i) a swab(e.g., a throat swab) for use in collecting a biological sample, (ii) anextraction chamber containing an extraction reagent, and (iii) a teststrip. In some embodiments, the swab is comprised of a stem and tip(absorptive component), where the tip incorporates a non-volatilereagent configured to extract, or aid in extracting, at least oneanalyte.

In some embodiments, the extraction solution formed by the dissolutionof the non-volatile reagent incorporated in the swab's tip in theextraction reagent serves to extract the analyte.

In some embodiments, the method of the present invention is comprisedof: (i) collecting a biological sample by a swab; (ii) transferring itto a chamber containing an extraction reagent, where this extractionreagent is complemented by an additional/second reagent incorporated inthe swab (e.g., but not limited to the tip of the swab), where theextraction reagent, when combined with the additional reagentincorporated in the swab, is configured to dissolve and/or react in-situwith the collected biological sample and extract the antigen/analytefrom the biological sample; (iii) contacting the extraction solutioncontaining the extracted analyte with a lateral flowimmunochromatographic assay device containing a labeled analyte-specificindicator; and (iv) determining the presence or absence of the extractedanalyte in the biological sample by measuring a signal (i.e., thepresence or absence of the signal) generated by a binding of a labeledanalyte-indicator complex to a capture reagent, where the capturereagent is immobilized on the lateral flow immunochromatographic assaydevice, and where the capture reagent is configured to specifically bindto the analyte.

In some embodiments, the present invention is a device for collectingand analyzing a biological or clinical sample, the device beingcomprised of (i) a swab, (ii) an extraction chamber containing anextraction reagent, and (iii) a test strip. In some embodiments, theswab is comprised of: (i) a stem made of a plastic or other material,which may be solid or porous, which is configured to provide mechanicalsupport for the swab tip and optionally also promote liquid transfer viacapillary flow through the stem's porous core; (ii) a tip at one end ofthe stem made of fiber or foam of polymeric material or other absorbentmaterial coated or spun or otherwise deposited on the stem's end (takentogether, stem and tip are termed: “swab”). In case the stem is madefrom porous material, the whole swab may be manufactured from the samefoam or other porous material thereby simplifying its manufacturingprocess, and (iii) a non-volatile reagent that comprises an essentialcomponent of the solution used to extract, or aid in extracting, thesample being collected by the swab and is (a) coated onto the swab stemin the area underlying its tip or (b) incorporated within the swab's tipabsorbent material. In some embodiments, the extraction chambercomprises an elongated tube having, for example, but not limited to: (i)a rounded, v-shaped, or flat bottom geometry and (2) a 4-10 mm innerdiameter, where the extraction chamber is configured to allowsubstantially simultaneous accommodation of both swab tip and teststrip. In some embodiments, the test strip is a conventional, lateralflow, immunochromatograpic assay device. In some embodiments, thenon-volatile material is a water-soluble organic acid. In someembodiments, the non-volatile material is a water-soluble inorganicacid. In some embodiments, the non-volatile material is an insolubleacidic polymer.

In some embodiments, the amount of the non-volatile acidic material isin the range of 50 and 800 micromoles. In some embodiments, the amountof the non-volatile acidic material is in the range of 100 and 800micromoles. In some embodiments, the amount of the non-volatile acidicmaterial is in the range of 150 and 800 micromoles. In some embodiments,the amount of the non-volatile acidic material is in the range of 200and 800 micromoles. In some embodiments, the amount of the non-volatileacidic material is in the range of 250 and 800 micromoles. In someembodiments, the amount of the non-volatile acidic material is in therange of 300 and 800 micromoles. In some embodiments, the amount of thenon-volatile acidic material is in the range of 350 and 800 micromoles.In some embodiments, the amount of the non-volatile acidic material isin the range of 400 and 800 micromoles. In some embodiments, the amountof the non-volatile acidic material is in the range of 450 and 800micromoles. In some embodiments, the amount of the non-volatile acidicmaterial is in the range of 500 and 800 micromoles. In some embodiments,the amount of the non-volatile acidic material is in the range of 550and 800 micromoles. In some embodiments, the amount of the non-volatileacidic material is in the range of 600 and 800 micromoles. In someembodiments, the amount of the non-volatile acidic material is in therange of 650 and 800 micromoles. In some embodiments, the amount of thenon-volatile acidic material is in the range of 700 and 800 micromoles.In some embodiments, the amount of the non-volatile acidic material isin the range of 750 and 800 micromoles.

In some embodiments, the amount of the non-volatile acidic material isin the range of 2 and 750 micromoles. In some embodiments, the amount ofthe non-volatile acidic material is in the range of 2 and 700micromoles. In some embodiments, the amount of the non-volatile acidicmaterial is in the range of 2 and 650 micromoles. In some embodiments,the amount of the non-volatile acidic material is in the range of 2 and600 micromoles. In some embodiments, the amount of the non-volatileacidic material is in the range of 2 and 550 micromoles. In someembodiments, the amount of the non-volatile acidic material is in therange of 2 and 500 micromoles. In some embodiments, the amount of thenon-volatile acidic material is in the range of 2 and 450 micromoles. Insome embodiments, the amount of the non-volatile acidic material is inthe range of 2 and 400 micromoles. In some embodiments, the amount ofthe non-volatile acidic material is in the range of 2 and 350micromoles. In some embodiments, the amount of the non-volatile acidicmaterial is in the range of 2 and 300 micromoles. In some embodiments,the amount of the non-volatile acidic material is in the range of 2 and250 micromoles. In some embodiments, the amount of the non-volatileacidic material is in the range of 2 and 200 micromoles. In someembodiments, the amount of the non-volatile acidic material is in therange of 2 and 150 micromoles. In some embodiments, the amount of thenon-volatile acidic material is in the range of 2 and 100 micromoles. Insome embodiments, the amount of the non-volatile acidic material is inthe range of 2 and 50 micromoles. In some embodiments, the amount of thenon-volatile acidic material is in the range of 2 and 10 micromoles. Insome embodiments, the amount of the non-volatile acidic material is inthe range of 5 and 10 micromoles. In some embodiments, the amount of thenon-volatile acidic material is in the range of 3 and 5 micromoles.

In some embodiments, the amount of the non-volatile acidic material isin the range of 30 and 200 micromoles. In some embodiments, the amountof the non-volatile acidic material is in the range of 50 and 200micromoles. In some embodiments, the amount of the non-volatile acidicmaterial is in the range of 100 and 200 micromoles. In some embodiments,the amount of the non-volatile acidic material is in the range of 150and 200 micromoles. In some embodiments, the amount of the non-volatileacidic material is in the range of 30 and 150 micromoles. In someembodiments, the amount of the non-volatile acidic material is in therange of 30 and 100 micromoles. In some embodiments, the amount of thenon-volatile acidic material is in the range of 30 and 50 micromoles.

In some embodiments, the swab and test strip are integrated into asingle device by contacting the back end of the swab tip (i.e., theabsorptive component) to the sample pad of the test strip allowing forcapillary liquid communication between both media.

In some embodiments, the present invention is a method for determiningthe presence or absence of an analyte in a sample, comprising thefollowing steps: (a) collecting a biological sample using a swabincorporating an non-volatile compound that comprises a component of thesample extraction solution, (b) extracting the analyte from the samplein an assay chamber containing an extraction reagent, (c) interactingthe extraction solution containing the extracted analyte with a lateralflow immunochromatographic assay test strip, by: (i) inserting this teststrip into the extraction solution or (ii) transferring the extractionsolution contents to the device by means of a valve, pipette or anyother common means of fluid transfer; (d) allowing the formation ofanalyte-labeled reagent complexes by the reaction of the analyte flowingthrough the lateral flow device with the analyte-specific labeledreagent carried by the device; (e) determining the presence or absenceof the analyte in the sample by the presence or absence of a signalformed by the binding of the analyte-labeled reagent complex to ananalyte-specific capture reagent immobilized at the lateral flow devicedetection zone. In some embodiments, the analyte is Strep A antigen. Insome embodiments, the extraction reagent is 0.2M to 5M nitrite saltsolution.

In some embodiments, the extraction reagent is 1M to 5M nitrite saltsolution. In some embodiments, the extraction reagent is 2M to 5Mnitrite salt solution. In some embodiments, the extraction reagent is 3Mto 5M nitrite salt solution. In some embodiments, the extraction reagentis 4M to 5M nitrite salt solution. In some embodiments, the extractionreagent is 0.2M to 4M nitrite salt solution. In some embodiments, theextraction reagent is 0.2M to 3M nitrite salt solution. In someembodiments, the extraction reagent is 0.2M to 2M nitrite salt solution.In some embodiments, the extraction reagent is 0.2M to 1 M nitrite saltsolution. In some embodiments, the extraction reagent is 1 M to 2Mnitrite salt solution. In some embodiments, the extraction reagent is1.5M to 2M nitrite salt solution. In some embodiments, the extractionreagent is 1 M to 1.5M nitrite salt solution. In some embodiments, theextraction reagent is 3M to 4M nitrite salt solution.

In some embodiments, the device of the present invention relates toimmunoassays involving Streptococcus Group A carbohydrate antigenextraction prior to assay performance by individuals typically lackingextensive training in laboratory techniques. In some embodiments, thedevice is configured to render transfer of the extracted sample to theimmunoassay device for analysis unnecessary. In some embodiments, thedevice of the present invention is configured to allow the use of a swabcarrying at least one analyte extraction solution component. In someembodiments, the at least one analyte extraction solution component is anon-volatile dry solid (e.g. citric acid), and is incorporated into theswab. In some embodiments, the device is configured to simplify thesteps of analyte extraction processing, by reducing the number ofreagent solutions and assay steps. In some embodiments, the amount of(a) non-volatile reagent immobilized on the swab and (b) extractionreagent in the extraction chamber are pre-aliquoted onto the device. Insome embodiments, the device of the present invention reduces errors dueto incorrect reagent dispensing and sample transfer steps. In someembodiments, device of the present invention is configured to detect thepresence or absence of Streptococcus Group A antigen in swab samples,e.g., throat swab samples.

Typically, the device and methods of the present invention allow for thedetection of analytes in samples which need to be extracted or otherwisechemically manipulated prior to assay performance, while simplifying thesample extraction process and reducing sample manipulation.

The device of the present invention is configured for collecting abiological or a clinical sample, the device being comprised of a stemmade of plastic or similar supportive material and a tip, located at oneend of the stem, made of fiber or foam or other absorbent materialcoated or spun or otherwise deposited on the stem's end (taken together,stem and tip constitute the “swab”). In some embodiments, depositednon-volatile material is either coated onto the swab stem in the areadirectly underlying its tip or incorporated within the stem tip'sabsorbent material. In some embodiments, the non-volatile material isconfigured for use in extracting and/or treating the biological/clinicalsample collected onto the swab.

In some embodiments, the present invention is a method for determiningthe presence or absence of Streptococcus Group A antigen in a sample,comprising the following steps: (a) collecting a biological sample usinga throat swab incorporating a non-volatile acid; (b) extracting theantigen from the sample in an assay chamber containing a extractionreagent, where the extraction reagent is a nitrite salt aqueoussolution, (c) interacting the extraction solution containing theextracted analyte (Strep A polysaccharide antigen) with a lateral flowimmunochromatographic assay device, where the interaction is allowed by(i) insertion of a lateral flow test strip into the extraction solutionor by (ii) transferring the extraction solution contents to the assaydevice by means of a valve, pipette, capillary forces, or any otherknown means of fluid transfer; (d) allowing the formation ofanalyte-labeled reagent complexes by the reaction of the analyte flowingthrough the lateral flow device with the labeled analyte-specificreagent carried by the lateral flow device; (e) determining the presenceor absence of the analyte in the sample by the presence or absence of asignal formed by the binding of the analyte-labeled reagent complex toan analyte-specific capture reagent immobilized at the lateral flowdevice detection zone, or any combination thereof.

In some embodiments, the present invention is a device, where the deviceis configured to allow conducting an immunoassay for extractedStreptococcus Group A carbohydrate antigens. In some embodiments, thetransfer of an extracted sample fluid to the immunoassay device foranalysis is not required. In some embodiments, contact between theextraction solution and the diagnostic strip is sufficient for assayperformance. In some embodiments, the lateral flow immunochromatographicassay methods of the present invention can be performed usingcommercially available test strips. In some embodiments, the device ofthe present invention is configured to use swabs carrying at least oneanalyte extraction solution component(s), wherein the analyte extractionreagent component can be in the form of a non-volatile dry solid appliedto the swab by means of incorporation. In some embodiments, the devicereduces analyte extraction process complexity and reduces the number ofrequired reagents and assay steps. In some embodiments, the device isconfigured to allow analyte extraction and analysis within a singlechamber using a single, pre-filled extraction reagent. In someembodiments, the device is configured to be used without the need foreither swab or extraction solution removal from the extraction chamberfor assay performance.

Examples

In order for the advantages of the invention to be readily understood, amore particular description of the disclosure briefly described abovewill be rendered by reference to specific embodiments that areillustrated in the appended drawing(s). It is noted that the drawings ofthe disclosure may not be to scale and are merely schematicrepresentations, not intended to portray specific parameters of thedisclosure. It is to be understood that these drawing(s) depict onlytypical embodiments of the disclosure and are not therefore to beconsidered to be limiting of its scope, the disclosure will be describedand explained with additional specificity and detail through the use ofthe accompanying drawing(s), in which:

FIGS. 1A-B shows an embodiment of the device of the present invention,including a swab, comprised of a stem (1) and a rounded tip made ofabsorbent material (2) for clinical sample collection, with the tipincorporating a non-volatile reagent essential for efficient analyteextraction from the sample. Reagent incorporation (3) can be performedby coating the swab's stem (FIG. 1A) or on the back end of the swab'stip at the tip-stem boundary, away from the sample collection zone (FIG.1B). It should be noted that other reagent incorporation schemes arepossible and the current illustrations merely serve as examples,depicting preferred incorporation sites.

FIG. 2 shows an embodiment of the device of the present invention, inwhich extraction of Strep A antigen is carried out by inserting themodified throat swab (1-2) carrying a clinical sample into an extractionchamber (4) containing a pre-aliquoted solution of nitrite salt(extraction reagent) (5). The acid incorporated within the swab tip (3)dissolves in and acidifies the nitrite salt solution, thereby creating asolution of nitrous acid (extraction solution). This process promotesextraction of the polysaccharide-based Strep A antigen, which is furtherenhanced by thoroughly mixing the swab tip (2) in the extractionsolution (5). The swab may then be kept in the extraction chamber (4)while the extraction solution containing the extracted analyte isbrought in contact with the lateral flow strip (6-9). This can beaccomplished by dipping the sample pad of the test strip (8) in theextraction solution without removing the swab from the extractionchamber. The buffer optionally incorporated into the sample pad of thetest strip acts to neutralize the analyte-carrying acidic extractionsolution. The extracted analyte travels by capillary forces from thetest strip's sample pad to the site on the strip where theanalyte-specific labeled reagent has been incorporated (9), therebysolubilizing this reagent and forming labeled analyte-reagent complexes.These complexes continue to flow through the test strip, driven bycapillary forces until they reach the capture zone (7) where they bindto an immobilized capture reagent (usually another analyte-specificreagent/antibody, specific for either the analyte (T) or the mobilelabeled reagent itself (C)). This binding event results in theaccumulation of the label at the capture zone, which is visuallydetectable or can be monitored by the use of a dedicated reader,providing an indication for the presence of the analyte in the sample(T) and to the test's proper performance (C). Excess extraction solutionis wicked by the adsorbent pad (6) located at the distal end of thelateral flow test strip, which drives capillary flow along the teststrip membrane. In some embodiments, the swab tip can serve threedistinct functions: (i) collecting a biological or clinical sample,e.g., bacterial colonies from a patient's throat; (ii) incorporatingswab tip-adsorbed non-volatile reagent; and (iii) co-transferring boththe biological/clinical sample and swab-incorporated non-volatilereagent to the extraction reagent to effect efficient analyteextraction.

In an embodiment, the extraction chamber's dimensions, geometry andoptical properties are selected to allow assay performance within theextraction chamber without transferring either the extraction fluid orthe extracted swab from the extraction chamber. Since swab tip diametersand test strip widths commonly range around 4 mm (e.g., but not limitedto, 3 mm-6 mm), an extraction chamber is configured to have an innerdiameter of 4-10 mm for the performance of both analyte extraction andimmunochromatographic assay. Typically, commercial swab tip's lengthrange is 10-30 mm and the volume of the extraction fluid shouldtherefore be adjusted accordingly to allow sufficient swab tip coverageby the extraction reagent to optimize analyte extraction. In anexemplary embodiment, using a 5 mm inner diameter cylindrical extractionchamber and a 15 mm tipped swab, the minimal extraction solution volumecan be 250 uL allowing sufficient swab tip coverage for optimal analyteextraction. In some embodiments, the device of the present invention canuse a typical extraction fluid volume present in commercially-availableStrep A tests of 250-500 uL, which can be compatible with performing theextraction and assay steps within a single chamber.

In some embodiments, extraction chamber's geometry can be cylindrical,rectangular or any other shape that properly accommodates both the swaband the test strip. In some embodiments, the device is comprised ofmaterials including glass and inert plastics (e.g., but not limited to,polypropylene, polyethylene, polyethylene terephthalate, polystyrene,polycarbonate, etc., or any combination thereof) that exhibit minimalbinding of the extracted analyte to be analyzed. In some embodiments,optically-transparent materials are used for extraction chamber wallsfor test results visualization. In some embodiments, extraction chamberlength can be adjusted to allow the results reading area on the teststrip to protrude out of the extraction chamber aperture to allowresults visualization (FIG. 2 ). In some embodiments, the extractionchamber bottom can be rounded, v-shaped, flat or any other shape that isconfigured to accommodate both the swab tip and test strip, either aloneor when present together. In some embodiments, the use of rounded orv-shaped geometries reduces the volume required for optimal swab tipcoverage. In some embodiments, the use of rounded or v-shaped geometriesimproves co-accommodation of the test strip and the swab tip (see FIG. 2). In some embodiments, the extraction chamber is capped allowing itspre-filling by an accurate amount of the extraction reagent, whichassists in transport of the diagnostic kit to the site where the test isto be conducted, and uncapping the extraction chamber immediately priorto introduction of the swab carrying the biological sample.

In another embodiment of the device of the present invention, the fluidcontents of the extraction chamber can be transferred for analysis to atest strip contained within a lateral flow device by means of a valve orany other common mean of gravitational or capillary fluid transfer.

FIG. 3 shows an embodiment of the present invention, where the swabcarrying the incorporated non-volatile reagent (1-3) and the test strip(6-9) are integrated into a single device by connecting the back end ofthe swab tip (2) to the sample pad of the test strip (8) allowing forcapillary liquid communication between both media. This integrateddevice unifies the swab and diagnostic strip allowing clinical samplecollection and analysis in a single device. This configuration reducesthe diagnostic procedure's complexity by removing the requirement fortest strip addition to the extraction chamber following analyteextraction (as depicted in FIG. 2 ). In this exemplary embodiment,introduction of the swab tip (2) into the extraction reagent (5) presentin the extraction chamber (4) results in wicking of the extractionreagent into the swab tip. This leads to solubilization of thenon-volatile reagent carried by the swab tip (3) into the extractionreagent solution, which in turn provides the optimal conditions forefficient in-situ extraction of the analyte present on the swab tip, andpossibly reduces the need for swab tip stirring in the extractionreagent solution to effect efficient analyte extraction. Extractedanalyte then travels by capillary forces from the swab's tip into thetest strip's sample pad (8), where it is optionally neutralized by abuffer incorporated into this area of the test strip. The extractedanalyte continues to travel by capillary forces from the test strip'ssample pad to the site on the strip where the analyte-specific labeledreagent has been incorporated (9), thereby solubilizing this reagentwhich reacts with the analyte forming labeled analyte-reagent complexes.These complexes continue to flow through the test strip by capillaryforces until they reach the capture zone (7) where the complexes canbind to an immobilized capture reagent (typically anotheranalyte-specific reagent/antibody, specific for either the analyte (T)or the labeled reagent itself (C)). In this embodiment, the unitedswab-test strip is sheathed in a casing (10) that acts both as a handle,replacing the swab's stem for efficient clinical sample collection, anda cassette for the test strip. A window in this casing (11), which maybe simply an opening or composed of, e.g., but not limited to,transparent plastic film, allows easy reading of test results. Thecasing (10) and the extraction chamber (4) are configured to match thecasing outer diameter to the inner diameter of the extraction chamber,thereby allowing the casing to fit into and optionally cap theextraction chamber. Such capping can be affected by plugging theextraction chamber by the casing or by means of a screw joint or anyother typical means of forming such junction. Such capping has the addedadvantage of sealing the extraction chamber and preventing spillage ofthe extraction reagent out of the extraction chamber. This alsosimplifies test disposal in one unit following results reading. In anembodiment, the swab tip can serve four functions: (i) collecting abiological or clinical sample, e.g., bacterial colonies from a patient'sthroat; (ii) incorporating swab tip-adsorbed non-volatile reagent thataids in analyte extraction; (iii) co-transferring of bothbiological/clinical sample and swab-incorporated non-volatile reagent tothe extraction reagent to form the extraction solution and effectefficient analyte extraction; and (iv) providing fluid conduit allowingefficient capillary wicking of the extracted analyte solution to thetest strip's sample pad for analysis.

FIG. 4A shows one integrated device configuration that enables swabtip-diagnostic strip integration involving a casing made up of twomatching, interlocking valves (10 a,b). One such valve—the cover (10a)—is configured to clasp the swab tip (2) via its stem (1), while theother valve—the base (10 b)—is configured to accommodate the test strip(6-9). These two interlocking valves are designed to bring the swab tip(2) and the test strip's sample pad (8) in physical contact upon joiningthese two valves. The results window (11) in the casing allows directvisualization of diagnostic test results. The casing cap (12) thatprotects the swab tip from mechanical damage is being removed to exposethe swab's tip immediately prior to clinical sample collection. FIG. 4Bdepicts further detail of the interaction between the casing cover (10a) and the swab stem (1), showing one possible clasping mechanisminvolving protrusions in the cover (13) that serve to firmly immobilizethe swab stem within the casing cover. FIG. 4C depicts such fullyassembled integrated device.

In some embodiments of the device of the present invention, theextraction reagent is made exclusively of solvent, e.g., but not limitedto, water. In other embodiments, the extraction reagent is an aqueoussolution, having nitrite salt concentration in the range of 0.2-5 M. Theextraction reagent may also contain any typically-used, non-ionic,detergent (e.g., but not limited to, NONIDET P-40, Tween-20 TritonX-100, CHAPS, or any combination thereof), a pH buffering compound(e.g., but not limited to phosphate, Tris, HEPES, or any combinationthereof), a chelating agent (e.g., but not limited to EDTA, EGTA, or anycombination thereof), a preservative (e.g., but not limited to,thimerosal, chlorhexidine digluconate, sodium benzoate, potassiumsorbate, sodium azide, sulfate salts of gentamicin, chloramphenicol andstreptomycin, protease inhibitors or phenolic compounds, or anycombination thereof), and a marker that changes the solution's color(e.g., but not limited to, from pink to light yellow) uponsolubilization of the additional reagent incorporated in the swab. Insome embodiments, analyte extraction from the sample is carried out bycontacting the throat swab with 400 uL of 1.2M sodium nitrite solutionpresent in the extraction chamber. In some embodiments, the swab tip isstirred in the extraction reagent by turning the swab against the sideof the tube. In some embodiments, after mixing the sample with thereagents, extraction is allowed to proceed for between 10 seconds and120 seconds, to allow for adequate polysaccharide antigen extraction. Insome embodiments, the extraction is allowed to proceed for between 10seconds and 60 seconds, to allow for adequate polysaccharide antigenextraction. In some embodiments, the extraction is allowed to proceedfor between 10 seconds and 30 seconds. In some embodiments, theextraction is allowed to proceed for between 30 seconds and 60 seconds.In some embodiments, the extraction is allowed to proceed for between 30seconds and 120 seconds. In an embodiment, the swab is left in theextraction chamber while the test strip is introduced to it (FIG. 2 ).In another embodiment (FIG. 3 ), the analyte is extracted in situ at theswab's tip by the incoming extraction reagent that becomes acidified bythe non-volatile acid incorporated in the swab's tip. In someembodiments, the configuration has the advantage of allowing additionalcontact time between the extraction reagent and the sample carried bythe swab tip which optimizes antigen extraction.

In some embodiments of the device of the present invention,neutralization of the nitrous acid solution following extraction of theantigens is affected by a buffer material incorporated within the samplereceiving portion (8) of the lateral flow immunochromatographic assaydevice (test strip). In an embodiment, analyte extraction and testing isperformed within the same chamber without swab or fluid removal,introduction of the test strip into the extraction chamber followinganalyte extraction causes the buffer contained within the test strip todissolve into the extraction solution thereby neutralizing the nitrousacid solution and allowing optimal lateral flow immunochromatographicassay performance.

In some embodiments of the device of the present invention, the amountof acid incorporated within the swab is between 2-800 micromoles. Aswill be apparent to a person knowledgeable in the art, the amount ofacid that needs to be incorporated in the swab tip to allow foreffective Strep A antigen extraction depends on several factors,including (but not limited to): i) degree of acid acidity (pKa), ii)acid solubilization rate iii) volume and concentration of the extractionreagent that is to be acidified, iv) assay performance scheme anddetails (with or without swab removal, or by using an integrated device,etc.). Each combination of the above factors needs to be empiricallytested to assure optimal Strep A antigen extraction. As guideline forsuch optimal extraction may serve the pH range of the buffer resultingfrom mixing the two individual reagent solutions contained in typicalrapid Strep A diagnostic tests, which is typically a pH from 4.5 to 4.7.In some embodiments, acid can be selected from edible or typically safe(GRAS) non-volatile organic acids, such as, but not limited to: citric,ascorbic, etc. In some embodiments, the acid allows the swab to be useddirectly in contact with the throat tissue. In some embodiments, theswab can be configured to have a depositing or coating of non-volatileacid in a location that does not come in direct contact with the throattissue, such as on the swab's stem in the area directly underlying itstip or incorporated within the stem tip's absorbent material away fromthe area that is configured to come into contact with the throat tissue(FIGS. 1A-B). In some embodiments, the non-volatile acid can beco-incorporated with other inert agents (e.g., dextrose) to improve aciddissolution into the extraction buffer. In some embodiments, thenon-volatile acid can be co-incorporated with other inert agents toimprove taste (e.g., sweeteners, artificial flavors), or add coloring(e.g., food colorant). In some embodiments, the non-volatile acid can beco-incorporated with a mucolytic agent, such as N-acetylcysteine, toreduce sample viscosity. In some embodiments, the deposited non-volatileacid can be polymeric (e.g., but not limited to, polystyrene sulfonicacid, Nafion, etc.) and insoluble in the extraction reagent. In someembodiments, an ionic exchange between the acidic polymer and theextraction reagent result in the acidification of the latter withoutdissolution of the acidic polymer.

In some embodiments, the present invention is a device including a swabused for biological sample collection, where the swab can comprise,e.g., but not limited to, a polyester fiber (or fiber of other polymericmaterial) coated swab, foam swab, comprising open-cell polyurethane(e.g., but not limited to, Becton Dickinson foam swabs) or a flockedswab (e.g., but not limited to, swabs made by Copan). In someembodiments, to increase assay sensitivity, swab tips are constructed toadsorb minimal amount of analyte and retain minimal fluid volume whencoupled to a test strip's sample pad, thereby maximizing sample transferto the test strip for analysis. In some embodiments, this is achieved byproper selection and optimization of the following parameters: swabtip's construction material (e.g., but not limited to, polyester fiber),tip fiber diameter (e.g., but not limited to, 5-20 micron), fibersurface properties (e.g., but not limited to, hydrophilic coating), tipporosity (e.g., but not limited to, a porosity of 70-90%), tip lengthand diameter (typically, but not limited to 15-20 mm and 4-6 mm,respectively), and tip sorptivity. In some embodiments, tip sorptivitycan be chosen to allow rapid and quantitative capillary fluid transferfrom the extraction solution to the swab tip and from the swab tip tothe test strip's sample pad. Such capillary fluid transfer may or maynot be counteracted by gravitational forces—i.e., the deviceincorporating the swab and test strip may operate in either the verticalor horizontal positions. The respective sorptivity and porosity of theswab tip, the test strip's sample pad, and the additional membranes thatare used to construct the lateral flow-based test strip need to beselected and/or adjusted to allow efficient and continuous liquid flowfrom the swab tip to the sample pad of the test strip and from there tothe rest of the test strip components to allow for optimal deviceoperation. Such selection and/or adjustment of device component'sporosity and sorptivity should take into account the intended use of thedevice in terms of its vertical vs. horizontal positioning, for example,but not limited to, increasing capillary forces acting within devicecomponents to counteract external gravitational forces in devicesdesigned to operate in the upright position.

In some embodiments, the swab can be prepared with a non-volatile acidby dipping, spraying, or otherwise dispersing a solution of such acid ina volatile solvent, followed by solvent evaporation. In someembodiments, when employing acidic polymers, one end of the swab stem(where the tip is to be constructed) can be pre-wrapped or coated with amembrane or film made of the acidic polymeric material prior to theapplication of the fiber (e.g., but not limited to, wool, cotton,polyester, or any other fibrous or otherwise absorbent material, e.g.,but not limited to, a sponge) or foam matrix that is to create the tip(FIG. 1 ). In some embodiments, such configuration avoids directphysical contact between the acidic polymer carried by the swab and thethroat tissue from which the biological sample is to be withdrawn fordiagnosis.

In some embodiments, the present invention is a one-stepimmunochromatographic assay device configured to usecommercially-available lateral-flow based test strips. In someembodiments, the immunochromatographic assay device can contain a samplereceiving region comprising a porous material, configured to allowlateral flow of a sample containing extracted analytes from the samplereceiving region to the analyte detection region. In some embodiments,the sample receiving region and the analyte detection region can bepresent on a single porous member, or may comprise at least two separateporous members in lateral flow contact. In some embodiments, the samplereceiving region can contain dry analyte-specific labeled reagents thatare solubilized by the extraction reagent allowing analyte-labeledreagent complex formation. In some embodiments, the labeledanalyte-reagent complexes are configured to flow through the device bycapillary forces until they reach the capture zone where they bind tothe immobilized capture reagent, typically another analyte-specificantibody (i.e., a binding event). In some embodiments, the binding eventis detectable visually or by the use of a dedicated reader by theaccumulation of the label at the capture zone, which is configure toprovide an indication of the presence of the analyte in the sample.

While a number of embodiments of the present invention have beendescribed, it is understood that these embodiments are illustrativeonly, and not restrictive, and that many modifications may becomeapparent to those of ordinary skill in the art. Further still, thevarious steps may be carried out in any desired order (and any desiredsteps may be added and/or any desired steps may be eliminated).

What is being claimed is: 1) A device, comprising: a swab comprising anabsorptive component attached to a stem, wherein the absorptivecomponent comprises a non-volatile reagent, an extraction chamberconfigured to receive the swab and position the absorptive component ofthe swab configured to be in fluid communication with an extractionreagent, wherein the extraction chamber is filled with an extractionreagent, a test strip configured to be brought in fluid communicationwith the extraction reagent following extraction of an analyte from thebiological sample, comprising: a sample receiving portion configured toaccept a sample, wherein the sample is a liquid sample, and wherein thesample receiving portion is configured to permit the movement of ananalyte in the liquid sample through the test strip by capillary action,a site on the strip where the analyte-specific labeled reagent has beenincorporated, wherein the analyte-specific labeled reagent is configuredto bind the analyte from the biological sample, and wherein theanalyte-specific labeled regent is comprised of a molecule that bindsthe analyte specifically and with high affinity and is further labeledwith a label that allows its detection, a capture portion configured toreceive the analyte from the biological sample and the analyte-specificlabeled reagent so as to result in displaying a positive or negativeresult at the completion of the assay, and an adsorbent pad attached tothe distal end of the test strip and configured to bind to an excessextraction reagent thereby allowing flow across the test strip. 2) Thedevice of claim 1, wherein the analyte is Streptococcus Group ACarbohydrate Antigen. 3) The device of claim 1, wherein the extractionreagent is a nitrite salt. 4) The device of claim 1, wherein theextraction reagent is 0.2-5M nitrite salt solution. 5) The device ofclaim 1, wherein the non-volatile reagent is acidic. 6) The device ofclaim 1, wherein the swab and test strip are configured to be in fluidcommunication, so as to result in capillary flow from the absorptivecomponent of the swab to a sample receiving portion of the test strip.7) The device of claim 1, wherein the stem may be solid, porous, or anycombination thereof. 8) The device of claim 1, wherein the stem isconfigured to provide: mechanical support for the absorptive componentof the swab, and capillary flow through a porous core of the stem. 9)The device of claim 1, wherein the absorptive component of the swab iscomposed of a fiber, foam of polymeric material, absorbent material, orany combination thereof. 10) The device of claim 5, wherein the acidicnon-volatile reagent is deposited at the absorptive component of theswab by: spraying, dipping, or dispersing the solvent containing theacidic non-volatile reagent onto the absorptive component of the swab,and evaporating the solvent. 11) The device of claim 5, wherein theamount of the acidic non-volatile reagent is between 2 and 800micromoles. 12) The device of claim 5, wherein the acidic non-volatilereagent is soluble in the extraction reagent. 13) The device of claim 5,wherein the acidic non-volatile reagent is insoluble in the extractionreagent. 14) The device of claim 13, wherein the acidic non-volatilereagent is configured to exchange protons with the extraction reagent.15) The device of claim 13, wherein the insoluble acidic non-volatilereagent is deposited on the swab in a region configured to attach to theabsorptive component, and wherein the insoluble acidic non-volatilereagent is deposited on the swab by coating with a membrane or film madeof a polymeric material prior to the application of the absorptivecomponent, so as to result in avoiding direct contact between the acidand the subject. 16) The device of claim 5, wherein the acidicnon-volatile reagent is organic. 17) The device of claim 5, wherein theacidic non-volatile reagent is inorganic. 18) The device of claim 1,wherein the device comprises: a first structural component configured toattach the absorptive component of the swab by the stem of the swab, asecond structural component configured to house the test strip, whereinthe absorptive component and a sample receiving portion of the teststrip are in liquid communication when joining the first structuralcomponent and the second structural component. 19) A method, comprising:(i) collecting a biological sample of a subject by use of a swabcomprising a first, non-volatile reagent; (ii) transferring the swab toa chamber containing an extraction reagent, so as to allow thenon-volatile reagent to react with the extraction reagent and result ingenerating an extraction solution; (iii) extracting an analyte from thebiological sample of the subject by the extraction solution; (iv)contacting the extraction solution containing the extracted analyte witha lateral flow immunochromatographic assay device comprising a labeledanalyte-specific reagent; and (v) determining a presence or absence ofthe extracted analyte in the biological sample of the subject, whereinthe binding of the analyte to a capture reagent specifically immobilizesthe labeled analyte-indicator complex on the lateral flowimmunochromatographic assay device, and wherein the capture reagent isconfigured to specifically bind to the analyte. 20) A method comprising:(i) collecting a biological sample of a subject by an absorptivecomponent of a swab, wherein the absorptive component of the swab isconfigured to be in physical contact with a sample receiving portion ofa lateral flow immunochromatographic assay test strip comprising alabeled analyte-specific reagent; (ii) transferring the absorptivecomponent of the swab to a chamber containing an extraction reagent,wherein an extraction solution is formed in-situ by the reaction of theextraction reagent with the second non-volatile reagent incorporated inthe absorptive component of the swab, and wherein the extractionsolution is configured to extract the biological sample of the subject;(iii) extracting an analyte from the biological sample; (iv) determiningthe presence or absence of the extracted analyte in the biologicalsample of the subject by measuring a signal generated by a binding of alabeled analyte-indicator complex to a capture reagent, wherein thecapture reagent is immobilized on the lateral flow immunochromatographicassay device, and wherein the capture reagent is configured tospecifically bind the analyte.